The present invention relates to chromatography and, more particularly, to chromatography media in the form of a coherent pellet which rapidly hydrates when combined with water to form a gel useful for chromatography applications.
Purification of substances by chromatography, particularly employing high throughput technologies, is increasingly popular in the area of life science research. Traditionally, purification has been performed using columns containing the chromatography media in gel (slurry) form consisting of discrete beads of the media uniformly dispersed in a selected aqueous buffer. Column-based techniques are desirable from the viewpoint of large binding capacity; however, limitations arise when high throughput is desired since only several columns can be accommodated simultaneously.
To increase throughput, microcentrifuge spin columns, using about 1 ml or less of gel volume, have been used. Speed is thereby increased as is the ability to perform multiple (xcx9c12-24) separations at one time. To achieve higher throughput with similar gel volume, the use of gel dispersed in microtiter-filter plates, such as available from Whatman, Polyfiltronics, has become popular. Using these plates 96 or more purifications can be performed simultaneously.
Several problems, however, are encountered when using microtiter filter plates. The chromatography media tends to settle during dispensing of gel into the discrete wells, thus making it difficult to equalize the volume of media in all wells, particularly when dispensing is performed automatically, e.g., robotically. Also, because the gel is in the form of an aqueous slurry in the wells, it can leak out of the xe2x80x9cdrip directorxe2x80x9d or spillover the side walls of the wells, particularly if the gel is dispensed into the wells prior to arrival in the laboratory. A further problem centers on the lack of stability of many gel slurries, especially at room temperature. This results in the inability to store large batches of the gel for extended periods of time for sequent use in multiple purifications and assays.
One final drawback encountered with the use of currently available chromatography gels is that the gel usually must be first washed and then subsequently equilibrated in the aqueous medium (buffer) chosen for the desired application. This, of course, necessitates additional procedural steps for the end user and, accordingly, is time consuming.
Now, however, in accordance with the present invention, there is provided chromatography media in a form which obviates the problems heretofore encountered as discussed above. The present invention concerns a pellet and method for its manufacture and use where the pellet is a chromatography media characterized by a coherent aggregate of distinct beads having a capacity to resist a force, as demonstrated by a Schleuinger Pharmatron hardness of at least about 2 Kilo Ponds, and capable of being rapidly hydrated on addition of water to form a gel.
In one form, the invention defines a material in the form of a practically dry pellet comprising an aggregate of distinct beads of a selected chromatography media. On addition of an aqueous medium, the pellet rapidly hydrates to form a chromatography gel wherein the beads are swollen and substantially uniformly dispersed in the aqueous phase. An additional important feature of the pellet of the present invention is that the aggregate of beads is coherent as characterized as having a Schleuinger Pharmatron hardness, determined as hereinafter described, of at least about 2 Kilo Ponds (KP). In an additional aspect of this invention, the chromatography media is cross-linked agarose, dextran, or an acrylamide/azlactone copolymer, preferably those varieties thereof which are considered highly cross-linked indicating that the media can resist collapse under high column pressure. Agarose, designated by the manufacturers as xe2x80x9cfastxe2x80x9d or xe2x80x9csuperxe2x80x9d flow, is representative of a highly cross-linked media.
Being essentially dry and coherent, pellets can be placed, or as hereinafter described formed in situ, in the wells of microtiter or microfilter plates, and the plates immediately used, stored or shipped for future use; the problems of leakage, spillage and stability encountered with otherwise preformed gels being eliminated. For use, one needs only add the appropriate aqueous medium and, since hydration occurs almost immediately, rapid assays and high throughputs can be achieved.
In accordance with a further aspect of the present invention, there is provided a method for the preparation of the pelletized chromatography media described above. The process involves placing a chosen bed volume of an aqueous gel containing beads of a chromatography media in the bottom of a receptacle so that the gel molds into the bottom of the receptacle to an observable depth. Thereafter, the gel is dried under conditions of time, temperature and humidity selected to provide a pellet which is coherent and has the capacity to resist a force and is capable of being rapidly hydrated on the addition of water to form a gel wherein the beads are swollen and substantially uniformly dispersed in the water phase.